Hunter T C, Mehra R K
Environmental Toxicology Graduate Program, University of California, Riverside 92521, USA.
J Inorg Biochem. 1998 Mar;69(4):293-303. doi: 10.1016/s0162-0134(98)00005-1.
A Candida glabrata cadmium-sensitive mutant partially defective in glutathione production and exhibiting a complete absence of phytochelatins was used to clone a gene required for Cd tolerance. Transformation of the Cd-sensitive mutant with a genomic library from the wild-type C. glabrata led to the cloning of a gene that restored Cd tolerance and formation of Cd-glutathione and Cd-phytochelatin complexes. The cloned gene showed high levels of nucleic acid and protein sequence homology to the HEM2 genes, encoding porphobilinogen synthases, from several sources. It was shown that the C, glabrata Cd-sensitive mutant indeed exhibited a significant reduction in porphobilinogen synthase levels. The cloned C. glabrata gene complemented a hem2 mutant of Saccharomyces cerevisiae and restored porphobilinogen synthase activity in the mutant. The Cd sensitive mutant predictably showed decreased levels of sulfite reductase that requires siroheme, a metabolite produced in the heme biosynthetic pathway. The addition of cysteine, but not methionine, increased glutathione levels and Cd tolerance of both the wild-type and the mutant strain. However, addition of hemin chloride and methionine together restored Cd tolerance indicating that heme was required for transsulfuration of homocysteine to cysteine.
一株光滑念珠菌镉敏感突变体用于克隆镉耐受性所需基因，该突变体在谷胱甘肽产生方面存在部分缺陷且完全缺乏植物螯合肽。用来自野生型光滑念珠菌的基因组文库转化该镉敏感突变体，导致克隆出一个恢复镉耐受性以及形成镉 - 谷胱甘肽和镉 - 植物螯合肽复合物的基因。克隆出的基因与来自多个来源的编码胆色素原合酶的HEM2基因在核酸和蛋白质序列上具有高度同源性。结果表明，光滑念珠菌镉敏感突变体的胆色素原合酶水平确实显著降低。克隆出的光滑念珠菌基因补充了酿酒酵母的hem2突变体，并恢复了该突变体中的胆色素原合酶活性。镉敏感突变体中需要 siroheme（血红素生物合成途径中产生的一种代谢物）的亚硫酸盐还原酶水平可预测地降低。添加半胱氨酸而非甲硫氨酸可提高野生型和突变株的谷胱甘肽水平及镉耐受性。然而，一起添加氯化血红素和甲硫氨酸可恢复镉耐受性，表明血红素是同型半胱氨酸转硫化生成半胱氨酸所必需的。
